This proposal describes a 5-year research and training program that will allow the applicant to achieve his goal of becoming an independent investigator conducting clinically relevant basic and translational research in the area of tuberculosis (TB) and mycobacterial pathogenesis. He will build on his background in basic science research, gaining knowledge and skills in microbiology, bacterial genetics and immunology. At the same time, he will undertake research focused on the role that bacterial efflux pumps play in mycobacterial virulence and antibiotic tolerance. A mentoring team based at Seattle Children's Research Institute, Seattle BioMed and the University of Washington, with broad expertise in mycobacterial pathogenesis, bacterial genetics and infectious diseases will guide the candidate's training. Research Plan: TB killed over one million people in 2012 despite decades of available, effective antibiotic regimens. Barriers to eradication of TB include that treatment requires many months of antibiotics and that incomplete treatment may promote development of drug resistant TB strains. Recent studies demonstrate that bacterial efflux pumps contribute to antibiotic tolerance and intracellular persistence of Mycobacterium tuberculosis (Mtb) and therefore to both these barriers to eradication. This project seeks to understand how Mtb and other mycobacteria employ efflux pumps to survive within host immune cells and develop antibiotic tolerance. Aim 1: Determine which mycobacterial efflux pumps are required for intracellular survival, virulence and macrophage induced antibiotic tolerance of mycobacteria. The candidate will screen a library of archived M. marinum mutants to identify efflux pumps are necessary for these phenomena. Aim 2: Determine if key transcription factors are required for the coordinated regulation of efflux pumps in mycobacteria. Based on recent work to uncover the transcriptional regulatory network of Mtb, the candidate will identify and test the role of candidate transcription factors in regulating efflux pump mediated tolerance and intracellular survival. Aim 3: Determine if the roles of efflux pumps and the transcriptional networks that control their expression are conserved between Mtb and nontuberculosis mycobacteria (NTM). The applicant will test whether findings in Mtb can be applied to NTM, which are problematic pathogens in certain populations. Identifying efflux pumps important in these processes will facilitate future development of efflux pump inhibitors as novel therapeutic agents for TB and other mycobacteria. The training component will help to establish the candidate as an independent investigator in TB and mycobacterial pathogenesis.